Tratamento individualizado para a rinite alérgica baseado nas principais manifestações clínicas nasais e dos níveis de histamina e leucotrieno D4

(1)

www.bjorl.org

Brazilian

Journal

of

OTORHINOLARYNGOLOGY

ORIGINAL

ARTICLE

Individualized

treatment

for

allergic

rhinitis

based

on

key

nasal

clinical

manifestations

combined

with

histamine

and

leukotriene

D4

levels

夽

Congxiang

Shen

1

_,

_Fang

_Chen

1

_,

_Huigang

_Wang,

_Xinyu

_Zhang,

_Guanxue

_Li,

_Zhong

_Wen

∗

DepartmentofOtorhinolaryngologyHeadandNeckSurgery,SouthernMedicalUniversity,ZhujiangHospital,Guangzhou,China

Received13January2018;accepted25September2018 Availableonline24October2018

KEYWORDS Allergicrhinitis; Clinicaltyping; Individualized treatment; Histamine; Leukotrienes Abstract

Introduction:Thetypesofallergicrhinitisareroughlyclassiﬁedbasedonthecausative anti-gens,diseasetypes,predilectiontime,andsymptomseverity.

Objective: Toexaminetheclinicaltypingandindividualizedtreatmentapproachforallergic rhinitis andtodeterminetheoptimaltreatment methodfor thisdisease usingvarious drug combinationtherapies.

Methods:Atotalof108participantswithallergicrhinitisweredividedintothreegroupsbased onsymptoms.Subsequently,eachgroupwasfurthercategorizedintofoursubgroupsbasedon themedicationsreceived.Theefﬁcacyofthetreatmentswasevaluatedusingthevisualanalog scale VASscoresofthe totalandindividualnasal symptoms,declineindex ofthesymptom score,histamineandleukotrienelevels,andmRNAandproteinexpressionlevelsofhistamine 1andcysteinylleukotriene1receptors.

Results:Loratadine+mometasonefuroateandloratadine+mometasonefuroate+montelukast signiﬁcantlyimprovedthesneezingsymptomandreducedthehistaminelevelscomparedwith theothercombinationtherapies(p<0.05).Meanwhile,montelukast+mometasonefuroateand montelukast+mometasone furoate+loratadine considerably improved the nasal obstruction symptomanddecreasedtheleukotrieneD4levelscomparedwiththeothercombination ther-apies(p<0.05).

夽 _Please_cite_this_article_as:_Shen_C,_Chen_F,_Wang_H,_Zhang_X,_Li_G,_Wen_Z._{Individualized}_treatment_for_allergic_rhinitis_based_on_key_nasal

clinicalmanifestationscombinedwithhistamineandleukotrieneD4levels.BrazJOtorhinolaryngol.2020;86:63---73.

∗_{Corresponding}_author.

E-mail:wenzhong60@163.com(Z.Wen).

1 _These_two_authors_contributed_equally_to_this_study.

PeerReviewundertheresponsibilityofAssociac¸ãoBrasileiradeOtorrinolaringologiaeCirurgiaCérvico-Facial. https://doi.org/10.1016/j.bjorl.2018.09.007

1808-8694/©2018AssociaçãoBrasileiradeOtorrinolaringologiaeCirurgiaCérvico-Facial.PublishedbyElsevierEditoraLtda.Thisisanopen accessarticleundertheCCBYlicense(http://creativecommons.org/licenses/by/4.0/).

(2)

Conclusion:Clinicalsymptomevaluationcombinedwithexperimentaldetectionofhistamine andleukotrienelevelscanbeanobjectiveandaccuratemethodtoclinicallyclassifytheallergic rhinitistypes.Furthermore,individualizedtreatmentbasedonallergicrhinitisclassiﬁcationcan resultinagoodtreatmentefﬁcacy.

© 2018 Associação Brasileira de Otorrinolaringologia e Cirurgia Cérvico-Facial. Published by Elsevier Editora Ltda. This is an open access article under the CC BY license (http:// creativecommons.org/licenses/by/4.0/).

PALAVRAS-CHAVE Rinitealérgica; Tipiﬁcac¸ãoclínica; Tratamento individualizado; Histamina; Leucotrienos

Tratamentoindividualizadoparaarinitealérgicabaseadonasprincipais manifestac¸õesclínicasnasaisedosníveisdehistaminaeleucotrienoD4 Resumo

Introdução: Arinitealérgicaébasicamenteclassificadadeacordocomosantígenoscausadores, tiposdedoença,peridiocidadeegravidadedossintomas.

Objetivo:Avaliarostiposclínicoseaabordagemterapêuticaindividualizadapara cadatipo derinite alérgica edeterminarométodo detratamentoideal utilizando váriasterapias de combinac¸ãodefármacos.

Método: Umtotal de 108 participantes comrinite alérgica foram divididos em três grupos combasenossintomas.Posteriormente,cadagrupofoi subsequentementecategorizado em quatrosubgruposcombasenosmedicamentosrecebidos.Aeﬁcáciadostratamentosfoiavaliada utilizandoosescoresdaescalavisualanalógicaEVAdossintomasnasaistotaiseindividualmente, índicededeclíniodoescoredesintomas,níveisdehistaminaeleucotrienoseníveisdeexpressão demRNAeproteínadosreceptoresdehistamina1ecisteinil-leucotrieno1.

Resultados: Asassociaçõesentreloratadina+furoatodemometasona,assimcomoade loratad-ina+furoato de mometasona+montelucaste melhoraram significativamente o sintoma de espirrose reduziramos níveis de histamina em comparação àsoutrasterapias combinadas (p<0,05).Poroutrolado,aassociaçãomontelucaste+furoatodemometasona,assimcomoa associaçãomontelucaste+furoatodemometasone+loratadinamelhoraramconsideravelmente osintomadeobstruçãonasalediminuíramosníveisdeleucotrienoD4emcomparaçãocomas outrascombinações(p<0,05).

Conclusão:Aavaliaçãoclínicadossintomascombinadacomadetecçãoexperimentaldosníveis dehistaminaeleucotrienopodeserummétodoobjetivoeprecisoparaclassificarclinicamente ostiposderinitealérgica.Alémdisso,otratamentoindividualizadobaseadonaclassificação darinitealérgicapoderesultarnoaumentodaeficáciadotratamento.

© 2018 Associação Brasileira de Otorrinolaringologia e Cirurgia Cérvico-Facial. Publicado por Elsevier Editora Ltda. Este é um artigo Open Access sob uma licença CC BY (http:// creativecommons.org/licenses/by/4.0/).

Introduction

Thedevelopmentofallergicrhinitis(AR)mainlyinvolvesa T-helper Type 2 pattern of mucosal cell hyperfunction,1,2 whichresultsinchronic inflammationofthenasalmucosa caused bythe release of aseries of inflammatory media-tors,primarilyhistamineandleukotriene.3_Recent_research demonstratedthatthereleaseofleukotrieneoccursinboth theearlyandlatephasesofhypersensitivityresponses.4_This findingledtochangesin thetreatment conceptof AR,in whichtheeffectsofantileukotrienetreatmentwerefound tobecomparabletothoseofantihistaminesandintranasal corticosteroids.Ingeneral,effectivemanagementforARis comprisedofallergenavoidance,increasedpatient aware-nessandeducation,pharmacotherapy,andallergen-specific immunotherapy,amongothers.5_Currently,_the_medications usedtoclinicallycontrolARareclassifiedintothreemajor types:second-generationantihistamines,antileukotrienes,

and intranasal corticosteroids. Various investigations and meta-analysesanalyzedtheeffectofdifferentcombinations using these three drugs on AR, and their results showed that drug combinations were more effective than single drug therapy.6---10 _However, _the _development _and evalua-tion of these treatment methods were still based on the participants’subjectivereportsofnasalsymptomsand qual-ity of life. Additionally, few objective laboratory indexes wereusedincombinationwiththeparticipants’subjective appraisalsinsomeresearch.Inthepresentstudy,weaimed to classify the AR patients into several groups depending on the primary nasal symptoms and quantitative detec-tionofthelevelsofinﬂammatorymediators(histamineand leukotriene D4) released during hypersensitivity reactions intheperipheralbloodandnasalsecretionstoobjectively presentthepracticalinvivoandinvitroresponsesof indi-vidualparticipants.Additionally,weaimedtodesignvarious drug combination treatment methods and determine the

(3)

optimaltreatmentmethodsthroughcomparisonofpre-and post-therapyimprovementsinnasalsymptomsandchanges inhistamineandleukotrieneD4levels,aswellasmRNAand proteinexpressionlevelsofHistamine1(H1)andCysteinyl Leukotriene1(CysLT1)receptors.Theﬁndingsofthisstudy willserveasabasisfortheexplorationofnewapproaches forindividualizedARtreatment.

Materials

and

methods

Studypopulationandclinicalparameters

Participants with AR were recruited from the outpatient departmentofZhujiangHospital,SouthernMedical Univer-sity (Guangzhou,China)from January2014 toJune 2015. Theparticipantswereselectedthroughmedicalhistoryand allergic symptom assessments based on the inclusion and exclusion criteria, which arepatterned after a previously reportedguideline.11

Theinclusioncriteriaareasfollows:(1)age18---65years, withahistoryofARforatleastoneyear;(2)atleastthree ofthefourmajorsymptoms(sneezing,clearnasaldischarge ﬂow,nasalobstruction,andnasalitching),withthese symp-toms having >0.5---1h one-day cumulativeattack timefor participantswithPerennialAR(PAR);(3)nasalmucosa(or eveneyelid)edemaandobstruction;and(4)positive reac-tiontoatleastoneoftheallergenskinpricktests,witha skinallergytestindexscorethatisnotmorethan++or+++. Meanwhile,theexclusioncriteriaincludethefollowing: (1) existence of respiratory tract infectionand infectious suborbital nasosinusitis; (2) presence of allergic asthma, withasthmaticattackduringthelast5years;(3)existence ofnon-AR(aconditioninwhichthecausativeallergenwas not conﬁrmed, e.g., vasomotor rhinitis); (4) presence of severenasalseptumdeviationornasalpolyps;(5)<4weeks ofantihistamine(e.g.,Claritin)andcorticosteroiddrug dis-continuation;(6)<6weeksofastemizolediscontinuationand <1weekwithdrawaltimeforotherantihistaminedrugs;(7) <1weekwithdrawaltimefornasalsuctionantihistamines, glucocorticoidnasalsprays,ornosedropdrugs;and(8)use ofmacrolideantibioticsand/orimidazoleantifungalagents. The study protocol was approved by the Ethics Com-mittee of Zhujiang Hospital, Southern Medical University, andallparticipantsprovidedwritteninformedconsent. Fur-thermore, this study was conducted in accordance with the regulations of the Clinical Research Ethics Commit-tee and World Medical Association and the principles of theDeclarationHelsinki(ClinicalTrialRegistrationnumber: 2013-EBYHK-004).

Studypopulationcharacteristicsandtreatment grouping

Atotalof108participants(62menand46women)witha meanageof37yearswereincludedinthisstudy.Thirty-six participants(20menand16women)withameanageof36 yearsunderwentphysicalassessmentinthephysical exami-nationcenterofZhujiangHospital,whowerethenenrolled ashealthycontrols.

Table1 SinglenasalsymptomVASscorebasedonseverity

ofsymptoms.

Symptom Domain Scale

Sneezinga _No_symptom ₀_(no_symptom)

3---5 1---2(mild)

6---10 3---6(moderate)

≥11 7---10(severe)

Nasal obstruction

Nosymptom 0(nosymptom)

Awarenessbutnottroubled 1---2(mild) Intermittentoralternation 3---6(moderate) Almostallmouthbreathing 7---10(severe) a _The_daily_number_of_continuous_sneezing.

The sneezing and nasal obstruction scores of all par-ticipants were obtained, which were used for treatment grouping,usingtheVisualAnalogueScale(VAS)scoresthat are based on symptom severity (Table 1). As shown in Fig. 1, all participants were classiﬁed into three groups basedonthesymptoms’VASscorecombinedwiththe his-tamineandleukotrieneD4levels.The108participantswere dividedintosneezing(33participants,31%),nasal obstruc-tion(37participants,34%),andmixed(38participants,35%) groups.Subsequently,thethreesymptomgroupswere fur-therdividedintofoursubgroupsbasedonthemedications theyreceived.In thisstudy,loratadine,montelukast, and mometasonefuroate wereusedtotreatthepatients with AR. The participants randomly received 10mg loratadine andmontelukast(onedoseperday)and50␮gmometasone furoate(onepressforeachnostrilandonedoseperday). DeterminationofhistamineandleukotrieneD4 levels

Thevenous blood andnasalsecretions of theparticipants withARand thoseofthehealthy controlswere extracted todeterminethehistamineandleukotrieneD4levelsusing double-antibody sandwich enzyme-linked immunosorbent assay. The enzyme immunoassay kit was purchased from ShanghaiBlueGeneBiotechCo.,Ltd.(Shanghai,China),and theassays wereperformed in accordancewiththe manu-facturer’sinstructionsusingaSynergyHTMultimodeReader (BioTek,Winooski,VT,USA).

GeneandproteinexpressionofH1andCysLT1 receptors

Theturbinate mucosaltissues ofparticipantswithARand those who underwent nasal septum surgery, who were included as healthy controls, were examined to char-acterize the gene and protein expressions of H1 and CysLT1receptors.Areal-timefluorescentquantitative Poly-merase Chain Reaction (PCR) was performed to analyze themRNAexpression ofH1 and CysLT1 receptors. Allthe oligonucleotides purified through high-performance liquid chromatographywerechemicallysynthesizedbyInvitrogen Biotechnology Co. Ltd.(Shanghai, China). The primersF1 (CCTGGATACCGCAGCTAGGA)andR1 (GCGGCGCAATACGAAT-GCCCC)wereusedfor18SrRNAamplification(GenBankID:

(4)

108 participants nasal obstruction score < 5 and sneezing score ≥ 5 Sneezing Group (33 participants, 31%) Lor atadine + Montelukast

+ mometasone furoate Lor

atadine + Montelukast

Lor

+ mometasone furoate

Montelukast

+ mometasone furoate Montelukast + mometasone furoate Montelukast + mometasone furoate Montelukast

Lor

atadine

atadine + mometasone furoate Lor atadine L Group (8 par ticipants) L+M Group (9 par ticipants) L+M+MF Group (10 par ticipants) M+MF Group (10 par ticipants) L+MF Group (9 par ticipants) L+M+MF Group (10 par ticipants) L+MF Group (9 par ticipants) M+MF Group (9 par ticipants) M Group (9 par ticipants) L+M+MF Group (9 par ticipants) M+MF Group (8 par ticipants) L+MF Group (8 par ticipants) Mixed Group (38 participants, 35%) Nasal obstruction Group (37 participants, 34%) nasal obstruction score ≥ 5 and sneezing score< 5 nasal obstruction score ≥ 5 and sneezing score≥ 5

Figure1 SchematicillustrationoftreatmentgroupingofARparticipants.

NR003286)asaninternalreference.Meanwhile,theprimers F2 (TACGGAGTGAGCGGAAGC) and R2 (GCAGGTAGAGGAT-GTTCATAGG) were used for HRH1 ampliﬁcation (GenBank ID: NM 000861.3). Furthermore, the primers F3 (GCCAT-GAGCTTTTTCCGGTG)and R3(TGATTGTCTTGTGGGGGCTC) were used for CysLT1 ampliﬁcation (GenBank ID: AF 119711.1). The reverse transcription reaction system for cDNAsynthesiswaspreparedbasedonthemanufacturer’s instruction for the use of GoScriptTM _Reverse

Transcrip-tionSystem.QuantitativePolymeraseChainReaction(qPCR) was performed using ABI7500 real-time qPCR Thermocy-cle Instrument (ABI, Vernon, CA, USA), with cDNA as a template of FastStart Universal SYBR Green Master (Rox) (Roche, Indianapolis, IN, USA) and ␤-actin as a con-trol, in accordance with the manufacturer’s instruction. The average threshold Cycle (Ct) values for the three PCR reactions were determined and calculated as fold induction over thecontrol samplesusing thecomparative Ct method to calculate the changes in gene expression level.

The protein expression of H1 and CysLT1 receptors wasexamined through Westernblot assay. Additionally, a semiquantitativechemiluminescence(CL)analysiswas con-ducted using the Image Lab software after a grayscale scanningoftheX-rayﬁlm.The averageCLforthreecases were determined and calculated as the relative CL over thecontrolsamplestocalculatethechangesintheprotein expressionlevel.The proteinexpressionof H1andCysLT1 receptorswerequantiﬁedthroughcomparisonswith␤-actin and␥-tubulin,respectively.

Clinicaltreatmentevaluation

The Total Nasal Symptom Score (TNSS) wascalculated as thesumof thescoresof thefournasalsymptoms (rhinor-rhea,nasalitching,nasalobstruction,andsneezing),which wereassessedusingafour-pointLikertscalebasedon symp-tomseverity(Table2),inaccordancewiththeprotocol of apreviousreport.11 _The_single_nasal_symptom_scores_were recordedthroughoutpatientserviceandtelephonesurveys ofparticipants,whichwereconductedontheconsultation day and 2,4, and8 weeks aftermedication therapy.The TotalNasalSymptom ScoreReducingIndex(TSSRI), sneez-ingscore ReducingIndex(RI), andnasalobstruction score RIwereusedtoevaluatetheefﬁcacyoftheclinical treat-ment, in which RI=(pre-treatment score−post-treatment score)/pre-treatmentscore.Additionally,thehistamineand leukotriene D4 levels in the peripheral blood and nasal secretions,togetherwiththechangesinthemRNAand pro-tein expression levels of H1 and CysLT1 receptors in the turbinatemucosatissueswerealsousedtoassesstheclinical curativeeffect.

Statisticalanalysis

The correlations between the singlenasal symptom score (sneezingandnasalobstruction scores)andhistamineand leukotriene D4 levels in the peripheral blood and nasal secretions were assessed usingPearson’s correlation test. Anindependentsamplettestwasconductedtodetermine

(5)

Table2 Assessmentscalebasedonseverityofsymptoms.

Symptom Domain Scale

Rhinorrheaa _No_symptom ₀ 3---5 1 6---10 2 ≥11 3 Sneezingb _No_symptom ₀ 3---5 1 6---10 2 ≥11 3 Nasal itch-ing Nosymptom 0 Intermittent 1

Formicationbutcanstandit 2

Formicationandcannotstandit 3

Nasal obstruction

Nosymptom 0

Awarenessbutnottroubled 1

Intermittentoralternation 2

Almostallmouthbreathing 3

a _The_daily_number_of_blowing_nose. b _The_daily_number_of_continuous_sneezing.

theRIofthesinglenasalsymptomscoreandhistamineand leukotrieneD4levelsbeforeandaftertreatmentand evalu-atethevalidityofthepre-andpost-medicationeffectsfor eachgroup. Furthermore,a pairedsample ttest was per-formedtocalculatetheTNSSRIoftheARparticipantsafter 2,4,and8weeksof treatment.Ap-value<0.05was con-sideredstatisticallysigniﬁcant.Alldatawerepresentedas ¯

A±S,where ¯AandSrefertothemeanvalueandstandard deviationofthemean,respectively.Allstatisticalanalyses wereperformed usingSPSSversion19.0(IBMCo.,Armonk, NY,USA).

Results

ClinicalcharacteristicsofARparticipantsfromthe threesymptomgroupsbeforetreatment

Fig. 2 presents the AR clinical status of the participants fromthesneezing(n=33),nasal(n=37),andmixed(n=38)

Symptom Score 5 0 Total Score Nasal Obstr uction Score Sneezing Score 10 15

Mixed Group 38 Case Nasal obstruction Group 37 Case Sneezing Group 33 Case

Figure2 Totalnasalsymptom scoreandsinglenasal symp-tomscoreofARparticipantsfromsneezinggroup(n=33),nasal group(n=37)andmixedgroup(n=38)beforetreatment.

groups before treatment based on the TNSS and sneez-ingandnasal obstruction scores. The TNSS parametersof thethreesymptomgroupswerenotstatisticallysignificant (p>0.05). The sneezing andmixed groups showed signifi-cantly higher sneezing scores than the nasal obstruction group (p<0.05). The nasal obstruction and mixed groups had significantly higher nasal obstruction scores than the sneezinggroup(p<0.05).Meanwhile, Table3displays the mean histamine and leukotriene D4 levels in the periph-eralbloodandnasalsecretionsoftheARparticipantsfrom thethreegroups.TheTNSSandhistamineandleukotriene D4 levels of the three symptom groups showed markedly positivecorrelations (p<0.001). Table 4presents the cor-relations between the single nasal symptom score and histamineandleukotrieneD4levelsintheperipheralblood andnasalsecretions.Apositive relationshipwasobserved between the sneezing VAS scores and histamine levels in the peripheral blood and nasal secretions of the AR par-ticipantsfromthe sneezing,nasalobstruction, and mixed groups (r=0.43/0.43, 0.60/0.61, and 0.83/0.82, respec-tively;p<0.05).Similarly,highlypositivecorrelationswere also found between the nasal obstruction VAS score and leukotriene D4 levels of the sneezing, nasal obstruction,

Table3 HistamineandleukotrieneD4levelsinperipheralbloodandnasalsecretionsofARparticipantsfromsneezinggroup

(n=33), nasal group (n=37) andmixedgroup (n=38)before treatmentand correlationsof total nasal symptom score and histamineandleukotrieneD4levelsinperipheralbloodandnasalsecretions.

Group Numberof

patients

Histaminelevels(ng/mL) LeukotrieneD4levels(ng/mL) Correlation(r) Inperipheral blood Innasal secretions Inperipheral blood Innasal secretions Hisa _LtD4b Sneezinggroup 33 8.61_±3.05 5.20_±1.90 0.225_±0.098 0.135_±0.068 0.98 0.98 Nasalcongestion group 37 4.76±2.54 2.88±1.50 0.444±0.190 0.266±0.113 0.99 0.97 Mixedgroup 38 11.82±3.04 7.11±1.88 0.495±0.143 0.297±0.107 0.99 0.99 a _p_<_0.001. b _p_<_0.001.

(6)

Table4 CorrelationsbetweenthesinglenasalsymptomscoreandhistamineandleukotrieneD4levelsintheperipheralblood andnasalsecretions.

Group Nasalsymptom r(p)

Histaminelevels LeukotrieneD4levels

Inperipheralblood Innasalsecretions Inperipheralblood Innasalsecretions Sneezinggroup Sneezing 0.43(0.01) 0.43(0.01) 0.05(0.74) 0.13(0.45)

Nasalobstruction 0.17(0.32) 0.19(0.28) 0.54(0.002) 0.50(0.002) Nasalcongestion

group

Sneezing 0.60(0.00) 0.61(0.00) 0.01(0.94) −0.10(0.57) Nasalobstruction −0.21(0.21) −0.27(0.11) 0.56(0.00) 0.55(0.00) Mixedgroup Sneezing 0.83(0.00) 0.82(0.00) −0.06(0.75) 0.18(0.30) Nasalobstruction _−0.11(0.53) 0.39(0.02) 0.61(0.00) 0.60(0.00)

andmixedgroups(r=0.54/0.50,0.56/0.55,and0.61/0.60, respectively;p<0.05).

ClinicaloutcomesofARparticipantsafter

medicationtherapybasedonthesymptomscores andhistamineandleukotrieneD4levels

Fig.3displaystheTNSSRIaftertreatmentofthesneezing, nasal obstruction, and mixed groups. For the sneez-ing group (Fig. 3A), the loratadine+mometasone furoate andloratadine+montelukast+mometasonefuroate combi-nation therapies had significantly lower TNSS than the loratadine monotherapy and montelukast+mometasone furoate combination therapy after a specific medication administrationperiod(p<0.05). Forthenasal obstruction group(Fig.3B),boththemontelukast+mometasonefuroate andloratadine+montelukast+mometasonefuroate combi-nationtherapieshadsignificantlylowerTNSSthanthe mon-telukastmonotherapyandloratadine+mometasonefuroate combination therapy (p<0.05). Meanwhile, for the mixed group(Fig.3C),theloratadine+montelukast+mometasone furoate combination therapy had significantly lower TNSS thantheloratadine+montelukast,loratadine+mometasone furoate, and montelukast+mometasone furoate combina-tiontherapies(p<0.05).

Fig.4 shows the RIof the various clinical parameters, such as sneezing and nasal obstruction scores and his-tamine and leukotriene D4 levels in the peripheral blood and nasal secretions, after treatment for the sneezing, nasal obstruction, and mixed groups. Briefly, for the sneezinggroup(Fig.4A),loratadine+mometasonefuroate and loratadine+montelukast+mometasone furoate com-bination therapies significantly improved the sneezing symptomandreducedthehistaminelevelsintheperipheral blood and nasal secretions compared with loratadine monotherapy (p<0.05) and montelukast+mometasone furoate combination therapy (p<0.05). Furthermore, both montelukast+mometasone furoate and lorata-dine+montelukast+mometasone furoate combination therapies significantly improved the nasal obstruction symptom and decreased the leukotriene D4 levels in the peripheralbloodandnasalsecretionscomparedwith lorata-dine monotherapy (p<0.01) and loratadine+mometasone

furoate combination therapy (p<0.05). For the nasal obstruction group (Fig. 4B), loratadine+mometasone furoate, montelukast+mometasone furoate, and lorata-dine+montelukast+mometasone furoate combination therapies significantly improved the sneezing symptom and reduced the histaminelevels in the peripheral blood and nasalsecretions comparedwithloratadine monother-apy (p<0.05). Moreover, both montelukast+mometasone furoateandloratadine+montelukast+mometasonefuroate combination therapies significantly improved the nasal obstructionsymptomandloweredtheleukotrieneD4levels in the peripheral blood and nasal secretions compared with montelukast monotherapy (p<0.05) and lorata-dine+mometasonefuroatecombination therapy(p<0.05). Forthemixedgroup(Fig.4C),bothloratadine+mometasone furoateandloratadine+montelukast+mometasonefuroate combination therapies significantly improved the sneez-ing symptom and reduced the histamine levels in the peripheral blood and nasal secretions compared with loratadine+montelukast and montelukast+mometasone furoate combination therapies (p<0.05). Additionally, both montelukast+mometasone furoate and lorata-dine+montelukast+mometasone furoate combination therapies significantly improved the nasal obstruction symptom and decreased the leukotriene D4 levels in the peripheral blood and nasal secretions compared with loratadine+montelukast and loratadine+mometasone furoatecombinationtherapies(p<0.05).

GeneandproteinexpressionsofH1andCysLT1 receptors

Fig. 5 presents the changes in the gene and protein expression levels before and after medication. The AR participants had signiﬁcantly lower mRNA and protein expression levelsof H1and CysLT1 receptors after medi-cation therapy than before treatment (p<0.05),although nostatisticallysigniﬁcantdifference(p>0.05)inthese lev-elswasobservedbetweentheARparticipantsandhealthy controls.

(7)

C

B

A

8 4 2 8 4 2 8 4 2

Time after medicine treatment (week)

0,0 0,2 0,4 0,6 0,8 1,0 1,2 1,4 0,0 0,2 0,4 0,6 0,8 1,0 1,2 1,4 0,0 0,2 0,4 0,6 0,8 1,0 1,2 1,4 L Group L+M+MF Group M+MF Group L+MF Group L+M Group L+M+MF Group M+MF Group L+MF Group M Group L+M+MF Group M+MF Group L+MF Group * * * * * * * * * * ** * * * * * * * Reducing Inde x of TNSS Reducing Inde x of TNSS Reducing Inde x of TNSS

Figure3 TotalNasal SymptomScoreReducingIndex(TNSSRI)ofARparticipantsfor(A)sneezinggroup,(B)nasal obstruction groupand(C)mixedgroupafter2,4and8weeksofmedication.

Discussion

The main AR clinical classiﬁcation method is based on the subjective clinical symptoms reported by patients with AR, such as nasal symptoms and quality of life. AR is classically subdivided into SeasonalAR (SAR), PAR, and mixedAR,12_although_new_{classiﬁcations,}_that_is,_persistent

and intermittent AR, were recently proposed.13 _These classificationsplayedasignificantroleinthedevelopment ofARtreatmentandpreventionapproach.However,these classifications mainly focuson subjective nasal symptoms reported by patients with AR, without considering the changesinthemaininflammatorymediatorlevels.Hence, the severity of the disease and its effect on the quality

(8)

0,0 SN SN His in PD His in NS _{LTD4 in PD} LTD4 in NS NO His in PD His in NS _{LTD4 in PD} LTD4 in NS NO SN His in PD His in NS _{LTD4 in PD} LTD4 in NS NO 0,2 0,4 0,6 0,8 1,0 1,2 1,4 0,0 0,2 0,4 0,6 0,8 1,0 1,2 1,4 0,0 0,2 0,4 0,6 0,8 1,0 1,2 1,4 Reducing Inde x Reducing Inde x Reducing Inde x L+M Group L+M+MF Group M+MF Group L+MF Group M Group Clinical Parameters Clinical Parameters Clinical Parameters L+M+MF Group M+MF Group L+MF Group L Group L+M+MF Group M+MF Group L+MF Group

C

B

A

** ** ** * * * * * * * * * * * * * * * * * * * * * * * * * * * * * *

Figure4 Reducingindex ofsneezing score,nasalobstructionscoreandlevelsofhistamine andleukotrieneD4 inperipheral bloodandnasalsecretionsofARparticipantsfor(A)sneezinggroup,(B)nasalobstructiongroupand(C)mixedgroupafter4weeks ofmedication.SN,standsforsneezingscore;NO, standsfornasalobstructionscore;HisinPD,standsforlevelofhistaminein peripheralblood,HisinNS,standsfor levelofhistamineinnasalsecretions, LTD4inPD,standsfor levelofleukotriene D4in peripheralblood;LTD4inNS,standsforlevelofleukotrieneD4innasalsecretions.

(9)

HRH1 mRNA HRH1 CysL T1R mRNA CysL T1R

C

β-actin HRH1 51 KDa 39 KDa 8 8 7 7 6 6 5 5 4 4 3 3 2 2 1 1 43 KDa 56 KDa γ-tubin CysLT1R

B

A

2,0 0,0 _0,0 0,5 0,5 1,0 1,0

1,5 Prior Treatment 8 Case Prior Treatment 3 Case

Healthy Controls 8 Case

Healthy Controls 2 Case Post Treatment 8 Case

Post Treatment 3 Case

F

old induction

The relativ

e CL of protein

1,5

Figure5 Detection ofgeneexpressionandprotein expressionlevelsofhistamineH1andleukotrieneCysLT1 receptorsofAR participantsbefore andafter medication. (A)QuantiﬁcationofmRNAlevelsofhistamineH1 andleukotriene CysLT1receptors analyzedbyqPCR.(B)QuantiﬁcationofproteinlevelsofhistamineH1andleukotrieneCysLT1receptorsanalyzedbyWesternblot. (C)WesternblotimagesofhistamineH1,leukotrieneCysLT1andcontrolproteins.Priortreatment:Lane1,2,3;posttreatment: Lanes4,5,6,andhealthycontrol:Lanes7,8._␤-Actinand_␥-tublinwererespectivelyusedascontrolforhistamineH1andleukotriene CysLT1receptorsinqPCRandWesternblot.

of life of patients withAR are not objectively reflected. New, accurate, and objective clinical typing for AR is necessary for the development of effective, economical, and individualized treatment. AR has been reclassified as mild/moderate---severe and intermittent/persistent depending onthe symptom duration based on the guide-linesof AllergicRhinitisandItsImpact onAsthma (ARIA)5 and those developed in China (2009 edition).14 _This clas-sification method takes into consideration the symptom onset and disease severity, which closely reflect the actual symptoms experienced by patients. Recently, the 2014 Practical Guideline for the Management of Aller-gic Rhinitis in Japan15 _has _{reclassified} _AR _into _sneezing and nasal obstruction types based on the main nasal symptoms experienced by patients with AR depending on the allergy mediators, which may be more appropriate whendeterminingthechoiceofclinicaltreatment.

In anattempt toprovide a new,economical, and indi-vidualized treatment choice for patients with AR, we first combined the main nasal symptom evaluation with experimental detection of the changes in histamine and leukotrieneD4levelsandtheirH1andLT1receptor expres-sionstoexploretheroleofclinicaltypingandindividualized treatment of AR. In this study, we divided all the parti-cipantsintothreegroups basedontheassessment results of the nasal symptoms and histamine and leukotriene D4 levels.Ourclassificationdepended onthe combination of nasal symptom evaluation and experimental detection of histamineandleukotrieneD4levelstoreflectthechanges in the actual symptoms of the participants, which is a morereasonableandeffectiveapproachindeterminingthe appropriatedrugtobeusedthanthepreviousmethods.

HistamineandleukotrieneD4arethemaininﬂammatory mediatorsthatareimplicatedinARpathogenesis.Inastudy

(10)

byVadasetal.16 _that_involved₄₁_participants_who experi-enced acute allergicreactions, which ranged from Grade 1(least severe) toGrade 3 (mostsevere), the proportion ofparticipantswithelevatedhistaminelevelsincreasedas the allergic reaction grade increase. A similar result was reﬂectedinastudybyMayeretal.,17 _which_demonstrated thathighlyelevatedhistaminelevelsmightserveasasign for the developmentof fatal anaphylaxis. Changes in the leukotrieneslevelsduringallergicreactionsweresimilarto thatofhistamine.Additionally,obviousevidencesexist sug-gesting that leukotrienes levels are increased in patients withARfollowingallergenexposure.18---20_Our_results_showed thatthehistamineandleukotrieneD4levelsinthe periph-eralbloodandnasalsecretionsofARparticipantsfromall groupswereconsistentlyhigherthanthoseofhealthy con-trols.Furthermore,thehistamineandleukotrieneD4levels arehighlyassociatedwiththechangesinthesneezingand nasal obstruction symptoms, which is consistent withthe resultsofprevious studies.18,21,22 _The_elevation_of _the his-taminelevelmaybethecausefortheincreasedvasodilation andvascularpermeabilityofthenasalmucosalglands,which resultedinsneezing.Ontheotherhand,theincreaseinthe leukotrieneD4levelmayresultinthedilation,hyperemia, and thickening of the nasal mucosa, which lead tonasal obstruction.

AccordingtotheguidelinesofARIA5_and_those_from_China (2009 edition),14 _the _therapeutic _agents _for _AR _are clas-sified into six categories: antihistamines, nasal steroids, leukotriene receptor antagonists, anti-cholinergic agents, mast cell stabilizers, and nasal decongestants. Out of all thesetherapeuticagents,second-generationantihistamines (loratadine)andnasalsteroidsaretheclassicfirst-line med-icationsfor AR treatment. Recently, leukotriene receptor antagonists (montelukast) have gained increasing atten-tion in clinical research because of their effect on AR pathogenesis.23,24 _At _present, _many _combination thera-pies, which include loratadine, montelukast, and nasal steroids, are proposed, and their specific clinical cura-tiveeffectsonARhavebeenevaluated.6,10,25---27_Our_results suggested that loratadine and its combination clearly improved thesneezingsymptom and significantlyreduced the histamine level, whereas montelukast and its combi-nationobviously improved the nasal obstruction symptom and significantly decreased the leukotriene D4 level. Our resultsarein agreementwith therecent clinical practice guidelines for AR, which highly recommends the use of second-generation antihistamines with less sedating side effects in the treatment of AR patients with sneezing and nasal itching as the primary symptoms.28 _The _use of combination therapy is also recommended so that a ‘‘comprehensive’’ blocking effectis achieved asmuch as possible.

Histamineandleukotrieneareconsideredastheprincipal inﬂammatorymediatorswithmultiplephysiologicaleffects, whichresultsfromthebindingof theirreceptors.17,29 His-taminereceptorsareclassiﬁedintofoursubtypes,namely, H1,H2,H3,andH4,whichbelongtotheG-ProteinCoupled Receptor(GPCR)familymembers.30 _A_study_performed _by Akdis et al.31 _showed _that _H1 _receptors _are _most _critical inmediatinghistaminereleaseinallergicdisease.Cysteinyl Leukotriene(CysLts)receptors havethreeendogenous lig-ands, namely, Leukotriene C4, D4, and E4 (LTE4), which

alsobelongtotheGPCR familyandareclassifiedintotwo types (CysLT1 and cysteinyl leukotriene [CysLT2]) based on theirsensitivity toCysLT1-selective antagonists.32 Pre-vious studies demonstrated that CysLts have a clear role in the occurrence of pathophysiological conditions, such asasthma,AR,andother nasalallergies.21,33,34_A_study _by Figueroa et al.35 _on _the _mRNA _and _protein _expression _of CysLT1 and CysLT2 receptors in inflammatory cells from nasal lavage samples collected from participants during activeSARdemonstratedthatCysLT1receptor-positivecells werehighlyresponsiblefor thedevelopmentof inflamma-tion, which indicates the potential of CysLT antagonists in treating both AR and asthma. In our study, AR parti-cipants had higher gene and protein expression levels of H1 and CysLT1 receptors than healthy controls. Addition-ally,significant reductionsin themRNAandproteinlevels of H1 and CysLT1 receptors were observed after 4 weeks of antihistamine and antileukotriene treatment, which is consistentwiththechangesintherelevantmediatorlevels. Notably,thegeneandproteinexpressionlevelsofAR parti-cipantswerenotstatisticallydifferent(p>0.05)fromthose ofhealthycontrolsaftermedicationtherapy.Allthesedata suggesttheefficacyofcombinationtherapies,which signi-ficantlyimproved thenasalsymptomsofparticipantswith AR.

Conclusions

In conclusion,clinical symptom evaluation combinedwith experimental detection of histamine and leukotriene lev-els is amore objectiveand accuratemethod toclinically classify AR than previous approaches. Furthermore, our resultsdemonstratedthattheuseofcombinationtherapies is effective and this ﬁnding opensnew strategies for the individualizedARtreatment.

Funding

This work was supported by grants from Science and Technology Program Project of Guangdong Province (S2011010003947).

Conﬂicts

of

interest

Theauthorsdeclarenoconﬂictsofinterest.

Acknowledgements

Theauthorswouldliketothanktheclinical providerswho providedsupportandcarefortheparticipantsandthe sub-jectswhodonatedtime,blood,andtissuesamples.

References

1.GalliSJ, TsaiM, PiliponskyAM. Thedevelopment ofallergic inﬂammation.Nature.2008;454:445---54.

2.Halim TY, Krauss RH, Sun AC, Takei F. Lung natural helper cells are a critical source of Th2 cell-type cytokines in protease allergen-induced airway inﬂammation. Immunity. 2012;36:451---63.

(11)

3.Okubo K, Okuda M. Time-course changes in nasal airway resistanceafterhousedustantigenchallenge:withspecial refe-rencetolatephaseresponse.AllergolInt.1998;47:225---32. 4.Barrett NA, Maekawa A, Rahman OM, Austen KF, Kanaoka

Y. Dectin-2 recognition of house dust mite triggers cys-teinylleukotriene generation bydendritic cells.J Immunol. 2009;182:1119---28.

5.BousquetJ,KhaltaevN,CruzAA,DenburgJ,FokkensWJ,Togias A,etal.,AllergicRhinitisanditsImpactonAsthma(ARIA)2008 update(incollaborationwiththeWorldHealthOrganization, GA(2)LENandAllerGen).Allergy.2008;63Suppl86:8---160. 6.Goh BS, Ismail MI, Husain S. Quality of life assessment in

patients with moderate to severe allergic rhinitis treated with montelukast and/or intranasal steroids: a randomised, double-blind, placebo-controlled study. J Laryngol Otol. 2014;128:242---8.

7.ErdoganBA, Sanli A, PaksoyM, Altin G, AydinS. Quality of lifein patients withpersistent allergic rhinitistreated with desloratadinemonotherapyordesloratadineplusmontelucast combination.KulakBurunBogazIhtisDerg.2014;24:217---24. 8.EsteitieR,deTineoM,NaclerioRM,BaroodyFM.Effectofthe

additionofmontelukasttoﬂuticasonepropionateforthe treat-mentofperennialallergicrhinitis.AnnAllergyAsthmaImmunol. 2010;105:155---61.

9.TatarEC,Surenoglu UA,OzdekA, SaylamG,Korkmaz H.The effectof combined medical treatment on quality of life in persistentallergicrhinitis.IndianJOtolaryngolHeadNeckSurg. 2013;65:333---7.

10.LuY, Yin M,ChengL. [Meta-analysis ofleukotriene receptor antagonistmontelukast inthetreatmentofallergicrhinitis]. ZhonghuaErBiYanHouTouJingWaiKeZaZhi.2014;49:659---67. 11.EditorialBoardofChineseJOtorhinolaryngolHeadand Neck SurgeryCOSoCMA.Diagnosticandtreatmentprinciplefor aller-gicrhinitisandarecommendedscheme.ZhonghuaErBiYanHou TouJingWaiKeZaZhi.2005;40:166---7.

12.CiprandiG, Cirillo I,Vizzaccaro A, Tosca M, PassalacquaG, PallestriniE,etal.Seasonalandperennialallergicrhinitis:is thisclassiﬁcationadherenttoreallife?Allergy.2005;60:882---7. 13.BousquetJ,SchunemannHJ,SamolinskiB,DemolyP, Baena-CagnaniCE,BachertC,etal.AllergicRhinitisanditsImpacton Asthma(ARIA):achievementsin10yearsandfutureneeds.J AllergyClinImmunol.2012;130:1049---62.

14.SubspecialtyGroup ofRhinology EBoCJoOHaNS, and Subspe-cialtyGroupofRhinology,SocietyofOtorhinolaryngologyHead andNeckSurgery,ChineseMedicalAssociation.Guidelinesfor diagnosisandtreatmentofallergicrhinitis(2009,Wuyishan). Zhonghua Er Bi Yan Hou Tou Jing Wai Ke Za Zhi. 2009;44: 977---8.

15.OkuboK, KuronoY, FujiedaS,Ogino S,UchioE,OdajimaH, etal.JapaneseGuidelineforAllergicRhinitis2014.AllergolInt. 2014;63:357---75.

16.Vadas P, Perelman B, Liss G. Platelet-activating factor, his-tamine,andtryptaselevelsinhumananaphylaxis.JAllergyClin Immunol.2013;131:144---9.

17.MayerDE,KrauskopfA,HemmerW,MoritzK,JarischR,Reiter C.Usefulnessofpostmortemdeterminationofserumtryptase,

histamineanddiamineoxidaseinthediagnosisoffatal anaphy-laxis.ForensicSciInt.2011;212:96---101.

18.OkudaM,WataseT,MezawaA,LiuCM.Theroleofleukotriene D4inallergicrhinitis.AnnAllergy.1988;60:537---40.

19.GauvreauGM,PlittJR,BaatjesA,MacGlashanDW.Expressionof functionalcysteinylleukotrienereceptorsbyhumanbasophils. JAllergyClinImmunol.2005;116:80---7.

20.Thivierge M, Turcotte S, Rola-Pleszczynski M, Stankova J. Enhancedcysteinyl-leukotriene type1 receptorexpressionin Tcellsfromhousedustmite-allergicindividualsfollowing stim-ulationwithDerp.JImmunolRes.2015;2015:384780. 21.HaberalI,CoreyJP. Theroleofleukotrienesinnasalallergy.

OtolaryngolHeadNeckSurg.2003;129:274---9.

22.CobanogluB, ToskalaE,Ural A,Cingi C.Role ofleukotriene antagonists and antihistamines in the treatment of allergic rhinitis.CurrAllergyAsthmaRep.2013;13:203---8.

23.RodrigoGJ,YanezA.Theroleofantileukotrienetherapyin sea-sonalallergicrhinitis:asystematicreviewofrandomizedtrials. AnnAllergyAsthmaImmunol.2006;96:779---86.

24.SantosCB,HanksC,McCannJ,LehmanEB,PrattE,CraigTJ.The roleofmontelukastonperennialallergicrhinitisandassociated sleep disturbancesand daytime somnolence.Allergy Asthma Proc.2008;29:140---5.

25.MeltzerEO,PhilipG,WeinsteinSF,LaForceCF,MaliceMP,Dass SB,etal.Montelukasteffectivelytreatsthenighttimeimpact ofseasonalallergicrhinitis.AmJRhinol.2005;19:591---8. 26.PinarE,EryigitO,OncelS,CalliC,YilmazO,YukselH.Efﬁcacy

ofnasalcorticosteroidsaloneorcombinedwithantihistamines or montelukast intreatment ofallergic rhinitis. Auris Nasus Larynx.2008;35:61---6.

27.Pullerits T, Praks L, Ristioja V, Lotvall J. Comparison of a nasal glucocorticoid, antileukotriene, and a combination of antileukotrieneandantihistamineinthetreatmentofseasonal allergicrhinitis.JAllergyClinImmunol.2002;109:949---55. 28.Seidman MD, Gurgel RK, Lin SY, Schwartz SR, Baroody FM,

BonnerJR,etal.Clinicalpracticeguideline: allergicrhinitis. OtolaryngolHeadNeckSurg.2015;152:S1---43.

29.BisgaardH.Pathophysiologyofthecysteinylleukotrienesand effectsofleukotrienereceptorantagonistsinasthma.Allergy. 2001;56:7---11.

30.SadekB,StarkH.Cherry-pickedligandsathistaminereceptor subtypes.Neuropharmacology.2016;106:56---73.

31.AkdisCA,BlaserK.Histamineintheimmuneregulationof aller-gicinﬂammation.JAllergyClinImmunol.2003;112:15---22. 32.CapraV.Molecularandfunctionalaspectsofhumancysteinyl

leukotrienereceptors.PharmacolRes.2004;50:1---11.

33.Salvi SS, Krishna MT, Sampson AP, Holgate ST. The anti-inﬂammatoryeffectsofleukotriene-modifyingdrugsandtheir useinasthma.Chest.2001;119:1533---46.

34.Drazen JM,Israel E,O’Byrne PM.Treatmentofasthma with drugs modifying the leukotriene pathway. New Engl J Med. 1999;340:197---206.

35.FigueroaDJ,BorishL,BaramkiD,PhilipG,AustinCP,EvansJF. Expressionofcysteinylleukotrienesyntheticandsignalling pro-teinsininﬂammatorycellsinactiveseasonalallergicrhinitis. ClinExpAllergy.2003;33:1380---8.